This invention relates to vectors which encode stable messenger RNA (mRNA) and methods of using such vectors. In particular, this invention relates to vectors which establish controlled expression of recombinant genes within a tissue at levels which are useful for gene therapy and other applications.
The regulation of gene expression can be related to the rate of metabolic breakdown of mRNA molecules. Chen et al., J. Bact., vol. 172, no. 8, pp. 4578-4586 (1990). Such decay rates of individual mRNA species effects steady state expression levels of the corresponding protein in the cytoplasm. Id. Thus, the rate at which a particular protein is made is directly proportional to the cytoplasmic level of the mRNA which encodes it.
Studies have measured the stabilities of different mRNAs. Regulatory polypeptide mRNAs such as transcription factors or cytokines are often highly unstable whereas mRNAs for housekeeping proteins or maternal mRNAs which must be stored until after oocyte fertilization tend to be stable. Braverman, G., Cell Vol. 88, pp. 5-6 (1987); Rosenthal, E. T., et al., J.M.B. Vol. 166, pp. 307-327 (1983). Studies have also shown skeletal muscle mRNA to be distributed into two populations with regard to stability. Medford et al., J. Biol. Chem., vol. 258, pp. 11063-11073 (1983). One mRNA population has a half life of less than four hours and the other population has a half life of seventeen to over fifty-four hours. Id.
Researchers have examined factors which enhance or degrade mRNA stability. For example, the presence of 5' 7-methylguanosine triphosphate cap structures as well as the 3' poly (A) tails have been analyzed as mRNA stabilizers against degradation. Berstein, P., et al., Trends Biochem. Sci. Vol. 14, pp. 373-377 (1989); Drummond, D. R. et al., Nucleic Acid Res. Vol. 13 pp. 7375-7394 (1985); Sachs, A., Curr. Op. Cell Biol., Vol. 2, pp. 1092-1098 (1990). Studies suggest that the poly (A) tail and the poly (A)-binding protein (pABP) interact in order to regulate mRNA stability. Berstein, P., et al., Mol. Cell. Biol. Vol. 9, pp. 659-670 (1989). Such studies show increased mRNA stability when poly (A) tails are present. Deadenylation of the poly (A) tail, however, does not necessarily result in destabilization. Berstein, P., et al., Trends Biochem. Sci., Vol. 14, pp. 373-377 (1989).
Elements specific to mRNAs have been identified which can either stabilize or destabilize the transcript. These mRNA elements are thought to interact with cytoplasmic transacting factors. Studies show the transferrin receptor mRNA appears to be stabilized when an ironresponsive element in the 3' untranslated region (3'UTR) of the mRNA is bound by a specific 90 kDa binding protein. Mullner, E. W., et al., Cell Vol. 58, pp. 373-382 (1989); Casey, J., et al., EMBO J, Vol. 8, pp. 3693-3699 (1989). Other stability determining cis-acting sequences are located in the 5' untranslated region (5'UTR) or the coding region of an mRNA. Jones, T. R., et al., Mol. Cell. Biol. Vol. 7, pp. 4513-4521 (1987); Shyu, A., et al., Genes Dev., Vol. 3, pp. 60-72 (1989); Wisdom, R., et al., P.N.A.S., Vol. 86, pp. 3574-3578 (1989).
Studies have also shown that 3' UTRs containing AU-rich sequences correlate with rapid mRNA degradation by causing destabilization of the RNA transcript. Caput, et al., P.N.A.S., vol. 83, pp. 1670-1674 (1986); Cleveland, et al., New. Biol., vol. 1, pp. 121-126 (1989). Transcripts from many transiently expressed eukaryotic genes, including lymphokine genes and protooncogenes (c-myc and c-fos) contain AU-rich sequences in their 3' UTRs and are rapidly degraded in the cytoplasm. Schuler, et al., Cell, vol. 55, pp. 1115-1122 (1988); Shaw, et al., Genes & Dev., vol. 3, pp. 60-72 (1989). These studies have shown that deletion of AU-rich sequences from the c-fos or c-myc 3'UTR confers stability upon transcripts produced from transfected constructs. Wilson, et al., Nature, vol. 336, pp. 396-399 (1988); Jones, et al., Mol. Cell. Biol., vol. 7, pp. 4513-4521 (1987).
In addition, studies have shown that introduction of a AU-rich sequence from the granulocyte-macrophage colony-stimulating factor (GM-CSF) 3'UTR into the 3'UTR of the rabbit .beta.-globin gene confers instability upon the otherwise stable .beta.-globin mRNA. Shaw, et al., Cell, vol. 46, pp. 659667 (1986). AU-rich sequences have also been detected in the 3'UTR of interleukin-2, tumor necrosis factor-.alpha. and Human B Cell Stimulatory Factor II. Bohjanen, Mol. Cell. Biol., vol. 11, no. 6, pp. 3228-3295 (1991); Tonouchi, et al., Bio. Biopph. Res. Com., vol. 163, no. 2, pp. 1056-1062 (1989).
Studies have also compared the sequence of UTRs in vertebrate skeletal-.alpha., cardiac-.alpha. and .beta.-actin mRNA. These studies revealed regions of high sequence homology within the 3'UTR portion of each of these actin isoformic mRNA. This homology is greater among the a-cardiac and skeletal actin isoforms than between the .alpha.-skeletal actin and gactin isoform mRNAs. Mayer, et al., Nucl. Acids Res., vol. 12, pp. 1087-1100 (1984); Ponte et al., Nucl. Acids Res., vol. 12, pp. 1687-1696 (1984); Chang et al., Nucl. Acids Res., vol. 13, pp. 1223-1237 (1985). In comparison, 3'UTR sequences of other vertebrate genes, such as those encoding insulin and prolactin, share common coding regions but usually contain divergent 3'UTRs. Mayer et al., supra, Ponte et al., supra.